The techniques of genetic engineering have been successfully applied to the pharmaceutical industry, resulting in a number of novel products. Increasingly, it has become apparent that the same technologies can be applied on a large scale to the production of enzymes of value to other industries. The benefits of achieving commercially useful processes through genetic engineering are expected to include cost savings in enzyme production, productions of enzymes in organisms generally recognized as safe which are suitable for food products, and specific genetic modifications at the genomic level to improve enzyme properties, such as thermal stability and performance characteristics, as well as those which would increase the ease with which the enzyme can be purified.
Glucose oxidase is the enzyme which catalyzes the oxidation of glucose to gluconic acid with the concomitant production of hydrogen peroxide. The enzyme has many industrial uses, including its use in desugaring eggs, in the removal of oxygen from beverages, moist food products, flavors, and hermetically sealed food packages, and in the detection and estimation of glucose in industrial solutions, and in body fluids such as blood and urine.
Glucose oxidase was first isolated from cells of Aspergillus niger by Muller [Biocehmische Zeitschrift (1928), 199, 136-170 and (1931), 232, 423-424], and was also extracted from A. niger by Franke and Deffner [Annalen der Chemie (1939), 541, 117-150]. The production of glucose oxidase from cells of species of Penicillium chrysogenum, Penicillium glaucum, Pencillium purpurogenum, Aspergillus niger and Aspergillus fumaricus, has been described by Baker, in U.S. Pat. No. 2,482,724. A method for preparing glucose oxidase in which glucose oxidaseproducing strains of the genera Aspergillus and Penicillium are cultivated in medium having a low carbohydrate content is described in U.S. Pat. No. 3,701,715. The enzyme from Aspergillus niger (A. niger) has been purified to a high degree of purity, and reportedly has a molecular weight of approximately 150,000, an isoelectric point of 4.2, and a flavin adenine dinucleotide (FAD) content of 2 FAD per mole. Pazur and Kleppe (1964), Biochemistry 3, 578-583. The amino acid composition of the enzyme from A. niger, as well as its identity as a glycoprotein are also known. Pazur et al. (1965), Arch. Biochem. Biophys. 111, 351-357. However, neither the amino acid sequence of glucose oxidase, nor the nucleotide sequence encoding it are known.
A problem with utilizing glucose oxidase isolated from its native source is that the organisms which produce the enzyme may have contaminants which are deleterious for certain uses of the desired protein. For example, glucose oxidase is used for the commercial preparation of foodstuffs. However, A. niger, which is a major source of commercially prepared enzyme, is highly allergenic, and is not approved for use in food. Moreover, stringent purification procedures may be relatively expensive since glucose oxidase is primarily an intracellular enzyme. These problems could be solved by producing glucose oxidase in recombinant systems.
Fungal enzymes have been expressed from recombinant vectors. Glucoamylase from Aspergillus [Innis et al (1985), Science 228, 21-26] and endoglucanase I from Trichoderma reesei [Van Arsdell et al (1987), Biotechnology 5, 60-64] have been expressed in Saccharomyces cerevisiae.